RNA-Seq analysis (BrainSpan atlas) demonstrates an abundance of GRIK1 exon 9 in the human brain. The heat map shows the presence of exon 9 (45 bp; ENSE00001313812) that codes for GluK1 ATD splice in different brain regions from the embryonic to adult stage. Exon 9 expression coincides with well-studied areas for GRIK1 gene like cerebellar cortex, visual cortex, etc. Various regions of the brain and the donor age are represented on the x-axis and y-axis respectively. The donor age has been abbreviated as pcw (post-conception weeks), mos (months), and yrs (years). The regions of the human brain are abbreviated as: DFC (dorsolateral prefrontal cortex), VFC (ventrolateral prefrontal cortex), MFC (anterior [rostral] cingulate [medial prefrontal] cortex), OFC (orbital frontal cortex), M1C (primary motor cortex area M1, area 4), M1C.S1C (primary motor-sensory cortex [samples]), PCx (parietal neocortex), S1C (primary somatosensory cortex (area S1, areas 3,1,2), IPC (posteroventral [inferior] parietal cortex), A1C (primary auditory cortex core), A1C (primary auditory cortex [core]), TCx (temporal neocortex), STC (posterior [caudal] superior temporal cortex, area 22c), ITC (inferolateral temporal cortex (area TEv, area 20), OCx (occipital neocortex), V1C (primary visual cortex (striate cortex, area V1/17), HIP (hippocampus), AMY (amygdaloid complex), LGE (lateral ganglionic eminence), MGE (medial ganglionic eminence), CGE (caudal ganglionic eminence), STR (striatum), DTH (dorsal thalamus), MD (mediodorsal nucleus of thalamus), URL (upper [rostral] rhombic lip), CB (cerebellum) and CBC (cerebellar cortex). Blue and red color indicate zero and maximum expression respectively.

ATD splice insert affects the gating properties of the GluK1-1a homomeric receptors. (A) Displays mean-weighted Tau (τDes) values for GluK1-1a wild-type (red) and GluK1-2a wild-type (green) in the presence of glutamate. The inset shows representative normalized traces for GluK1-1a and GluK1-2a with 10 mM glutamate. (B) Displays the percent desensitization values calculated at 1 s for GluK1-1a wild-type (red) and GluK1-2a wild-type (green) in the presence of kainate. Representative normalized traces for GluK1-1a and GluK1-2a with 1 mM kainate are shown. (C) Demonstrates glutamate dose-response curves for GluK1-1a and GluK1-2a. Representative aligned traces for both receptors at various glutamate concentrations are shown. The kainate dose responses for the splice variants are shown in Figure 2-supplement figure 1. (D) The ratio of currents evoked by kainate and glutamate is plotted for GluK1-1a and GluK1-2a. (E) The ratio of currents evoked by the application of 10 mM glutamate at +90 mV and -90 mV for the GluK1-1a and GluK1-2a receptors is shown. Representative IV plots are depicted for GluK1-1a and GluK1-2a for the entire voltage ramp (-90 to +90 mV). Error bars indicate mean ± SEM, N in each bar represents the number of cells used for analysis, and * indicates the significance at a 95% confidence interval.

Whole-Cell patch clamp recordings of GluK1-1a, GluK1-1aEM, GluK1-2a, and GluK1-1a mutants in absence or presence of Neto1 (green) or Neto2 (peach).

Errors are reported as SEM. Statistical significance is reported at 95 % CI. P<0.05 (*), P<0.01 (**), P<0.001 (***), P<0.0001 (****) for comparisons between wild-type GluK1-1a receptor with EM construct, GluK1-2a, or various mutants in presence or absence of Neto proteins.

‘a’ denotes the lack of rectification index value due to no conductance observed at positive potentials in the K368-E mutant alone.

ATD splice affects the functional modulation of GluK1 kainate receptors by Neto proteins. (A) Shows mean-weighted Tau (τDes) values calculated at 100 ms for GluK1-1a (red) and GluK1-2a (green), respectively, with full-length Neto1 (blue/light blue) or Neto2 (black/gray), in the presence of glutamate. Representative normalized traces are shown for 100 ms application of 10 mM glutamate for HEK293 cells co-expressing GluK1-1a or GluK1-2a with Neto1 and Neto2. (B) Shows Tau (τRecovery) values plotted for GluK1-1a and GluK-2a, respectively, with full-length Neto1 or Neto2. Relative amplitude graphs for each receptor in the absence or presence of Neto proteins are also depicted. (C) Demonstrates the glutamate dose-response curves for GluK1-1a with Neto proteins. (D) Indicates the ratio of peak amplitudes evoked in the presence of 1 mM kainate and 10 mM glutamate for GluK1-1a or GluK1-2a with or without Neto proteins. (E) The ratio of currents evoked by the application of 10 mM glutamate at +90 mV and -90 mV for the receptors in the absence or presence of Neto proteins is shown. (F) Shows representative IV plots for GluK1-1a and GluK1-2a for the receptor alone versus with Neto proteins, respectively. Panels G and H show data recorded from outside-out pulled-patches. (G) Displays desensitization kinetics for GluK1-1a (red) and GluK1-2a (green) with or without Neto proteins, respectively. (H) Shows deactivation kinetics at 1ms for GluK1-1a (red) and GluK1-2a (green) with or without Neto proteins. Error bars indicate mean ± SEM, N in each bar represents the number of cells used for analysis, and * indicates the significance at a 95% confidence interval.

Excised patch outside-out electrophysiology of GluK1-1a and GluK1-2a in absence or presence of Neto1 (green) or Neto2 (peach).

Errors are reported as SEM. Statistical significance is reported at 95 % CI. P<0.05 (*), P<0.01 (**), P<0.001 (***), P<0.0001 (****) for comparisons between GluK1-1a and GluK1-2a receptors in presence of Neto proteins. Red p-values are statistical significance at 95% for comparison between GluK1-1a and GluK1-2a with either of the Neto proteins.

Mutation of GluK1-1a splice insert residues affects the desensitization and recovery kinetics of the receptor. Bar graphs (mean ± SEM) show a comparison between wild-type and mutant receptors for different kinetic properties. (A) Schematic representation of 15 residues ATD splice (K368ASGEVSKHLYKVWK382) in wild-type and mutant receptors under study (B) Mean-weighted Tau (τDes) values for GluK1-1a wild-type and mutant receptors in the presence of 10 mM glutamate. (C) Tau (τRecovery) recovery values for GluK1-1a and mutants. (D) The ratio of the peak amplitudes evoked in the presence of 1 mM kainate and 10 mM glutamate is shown for GluK1-1a mutants.(E) The rectification index represented by the ratio of currents evoked by 10 mM glutamate application at +90 mV and - 90 mV for the wild-type and mutant receptors is shown. The wild-type GluK1 splice variant data is the same as from Figure 2A and is replotted here for comparison. Error bars indicate mean ± SEM, N in each bar represents the number of cells used for analysis, and * indicates the significance at a 95% confidence interval.

Mutation of GluK1-1a splice insert residues affects the receptor modulation by Neto proteins. Bar graphs (mean ± SEM) show a comparison between wild-type and mutant receptors with Neto proteins for different kinetic properties. (A) Mean-weighted Tau (τDes) values for GluK1-1a wild-type and mutant receptors in the presence of 10 mM glutamate and expressed with Neto1/2. (B) Tau (τRecovery) recovery values for GluK1-1a and mutants with Neto1/2. (C) The ratio of the peak amplitudes evoked in the presence of 1 mM kainate and 10 mM glutamate for GluK1-1a mutants co-expressed with Neto1/2 is shown. (D) The rectification index represented by the ratio of currents evoked by 10 mM glutamate application at +90 mV and -90 mV for the wild-type and mutant receptors with Neto proteins is shown. The wild-type GluK1 splice variants’ data is the same as in Figure 2 and is replotted here for comparison. Error bars indicate mean ± SEM, N in each bar represents the number of cells used for analysis, and * indicates the significance at a 95% confidence interval.

Architecture of GluK1-1aEM reconstituted in nanodisc for SYM-bound desensitized state. (A) Shows the segmented density map colored according to unique chains of the receptor tetramer (A-blue, B-pink, C-green, and D-gold) at 5.23 Å in side view and 90° rotated orientations. (B) Shows the final model fitted in the EM map. (C & D) Top views of ATD and LBD layers. (E & F) Display the segmented map fitted with the corresponding distal (A & C) and proximal (B & D) chains. Receptor sub-domains, the position of splice insertion, and linkers are indicated.